Many cytoplasmic events are known to be regulated by one or more phosphorylation-dephosphorylation reactions. A primary event in the transformation of chick embryo fibroblasts by Rous sarcoma virus (RSV-CEF) may be the production of a 60K protein possessing phospho-transferase activity (Collett and Erikson, 1978). Recent evidence shows the pp60src to be concentrated on the cytoplasmic surface of the membrane (Willingham et al., 1979). Our preliminary work has identified two subsets of membrane phosphoproteins and one subset of membrane-associated phosphoprotein showing phosphorylation differences between normal and RSV-transformed chick embryo fibroblasts. The identification and function of these target proteins are not yet clear, nor has the phosphokinase activity of the isolated membranes been fully characterized. We propose to compare and characterize the endogenous phosphorylating ability of isolated membranes from normal and transformed cells. The assay procedure will be characterized in terms of the total incorporation of phosphate, pH optimum, substrate specificity, the influence of several activators, and product analysis by two-dimensional electrophoresis. Later, column chromatography of solubilized membrane proteins may lead to the isolation of a spectrum of kinase activity, the pattern of which may differ in transformed cells. We will continue to work on the phosphorylation of intact normal and transformed CEF cells with exogenously added 32Pi, comparing the labeling pattern in these membranes to isolated membranes which have been endogenously labeled with gamma-32P-ATP. Phosphoproteins will be analyzed by two-dimensional gel electrophoresis. Maintenance of topological relationships may direct the phosphorylaton of substrate proteins and a comparison such as this should help to assess the importance of such relationships. Our preliminary findings indicate that this area may be a very fruitful field for future research as distinct labeling patterns are seen.